Integrating Multiple Omics Identifies Phaeoacremonium rubrigenum Acting as Aquilaria sinensis Marker Fungus to Promote Agarwood Sesquiterpene Accumulation by Inducing Plant Host Phosphorylation

ABSTRACT The present study aimed to explore the factors that promote persistent agarwood accumulation. To this end, we first investigated the morphological changes and volatile compound distribution in five layers of “Guan Xiang” agarwood. The agarwood-normal transition layer (TL), an essential layer of persistent agarwood accumulation, showed clear metabolic differences by microscopy and GC-MS analysis. Microbiome analysis revealed that Phaeocremonium rubrigenum was the predominant biomarker fungus in the TL of “Guan Xiang” agarwood samples. Among the seven isolated fungi, P. rubrigenum exhibited a significantly heightened ability to induce the production in Aquilaria sinensis seedlings, especially for sesquiterpene. Tracing the proteome profile changes in P. rubrigenum-induced A. sinensis calli for 18 ds showed that the fungus-induced sesquiterpene biosynthesis increased mainly through the mevalonate (MVA) pathway. Specifically, the phosphorylation modification level, instead of the protein abundance of transcription factors (TFs), showed corresponding changes during sesquiterpene biosynthesis, thus indicating that induced phosphorylation is the key reason for enhanced sesquiterpene production. IMPORTANCE Agarwood is an expensive resinous portion derived from Aquilaria plants and has been widely used as medicine, incense, and perfume. The factors involved in steady agarwood accumulation remain elusive. Our current study suggests that as a TL marker fungus, P. rubrigenum could persistently promote agarwood sesquiterpene accumulation by inducing phosphorylation of the TFs-MVA network in A. sinensis. Moreover, our work provides strategies to improve agarwood industry management and sheds light on the potential molecular mechanisms of plant adaptation to native microbial conditions.

_Description of the different panels in Fig. 2 must be improved. This figure includes many data, that would be relevant for conclusions in the article. I found panels D unnecessary and could be removed. Panel A requires a more detailed explanation. Not clear what are the differences between the different graphs in this panel. Thus, it is difficult to reach a conclusion. _Authors need to revise the English to correct some misspelling words.
Reviewer #2 (Comments for the Author): In this manuscript, Liu et al. carried out extensive -omics-based studies to understand the interactions between Pm. rubrigenum and A. sinensis. The study itself is interesting to some extent but needs to be significantly developed before it can be accepted by Microbiology Spectrum. There are some places in the manuscript that are confusing to read and need further polishing.
A key question that was not answered in this manuscript, but is generally critical in the plant-microbe interactions, is to determine the compounds of interest are synthesized by which party. The authors have carried out extensive omics studies to show that sesquiterpene production is enhanced in A. sinensis, but it will also be helpful to investigate if these terpene-related genes are in Pm. rubrigenum. There are two potential scenarios that could be interesting: i) Pm. rubrigenum provides precursors to facilitate the production of sesquiterepen in A. sinensis, or ii) Pm. rubrigenum contains the terpene synthase genes. Therefore, in Line 185-190, The control experiment to conclude that Pm. rubrigenum was key to sesquiterpene production was not conclusive. It is likely that Pm. rubrigenum itself may produce these sesquiterpenes in the absence of A. sinensis seedlings.
Line 146, it is unclear how metabolism in TL changed a lot, is it a comparison with TL at a different time point or with AL and NL? Since no nuclei were observed in AL, the fungi species detected in AL do not represent the ones when AL cells were alive.
In Section 2.4, the proteome analysis of A. sinensis: it is unclear how the sampling of A. sinensis was carried out. Was A. sinensis one-year seedlings? Which layer (AL, NL, or TL) was examined with proteomics?
There are some grammatical errors throughout the manuscript and are not limited to the following: Line 30, "concerning" should be "for"; Line 36 "sesquiterpene promotion" should be "enhanced sesquiterpene production"; Line 135, "furtherly" should be "further". Staff Comments:

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Reviewers' comments and point-to-point response: Reviewer #1 (Comments for the Author):
Article "Integrating multiple omics identifies Phaeoacremonium rubrigenum acting as Aqularia sinensis marker fungus to promote agarwood sesquiterpene accumulation by inducing plant host phosphorylation" by Juan Liu and coworkers described a very detailed analysis trying to elucidate the mechanisms of agarwood formation by Aquilaria sinensis in interaction with the fungal microbiota. This work was made with Aquilaria sinensis host trees of the Guan Xiang area and detected P.
rubrigenum as an important promoter agent of agarwood sesquiterpene accumulation, which is one of the two major components of agarwood.
This manuscript is well written and clearly presented. It includes a deep analysis of sesquiterpenes detected in the five wood layers, and the relevance of the TL layer for this accumulation. They also found that Phaeoacremonium rubrigenum is one of the most abundant species in AL and TL. Authors also describe the secondary metabolites found in the different layer, the predominant fungi found in the different layers, as well as the sesquiterpenes found in them and described some interesting molecular features related with those properties (gene expression, protein phosphorylation, ...). Despite these previous considerations, authors can be required to improve clarity in the presentation of some data, and some figures could be shortened to reach this objective.
Thank you for the reviewer's advice. We have carefully polished our manuscript, and Figure 2 have been shortened to improve the presentation of our data.
Some minor points to be improved are included below, 1. Abstract Authors use an unusual way to abbreviate Phaeoacremonium as Pm., when the canonical way would be just a P. Thank you for the reviewer's advice. We have reviewed Pm. as P. in the whole manuscript. showing the distribution pattern of starch grains, polysaccharides, brown resin-like materials and live cells. AL, the agarwood layer; TL, the agarwood-normal transition layer; NL, the normal layer. I section, partial DL and partial DA; II section, partial AL and partial TL; III section, TL; IV section, NL. A-1 shows the brown resin-like materials and starch grains in I section by using I2-KI staining; A-2 shows the brown resin-like materials and polysaccharides in I section by using Periodic Acid Schiff (PAS) staining; A-3 shows the brown resin-like materials and nuclei in I section by using DAPI staining; B-1 shows the brown resin-like materials and starch grains in II section by using I2-KI staining; B-2 shows the brown resin-like materials and polysaccharides in II section by using Periodic Acid Schiff (PAS) staining; B-3 shows the brown resinlike materials and nuclei in II section by using DAPI staining; C-1 shows the brown resin-like materials and starch grains in III section by using I2-KI staining; C-2 shows the brown resin-like materials and polysaccharides in III section by using PAS staining; C-3 shows the brown resin-like materials and nuclei in III section by using DAPI staining; D-1 shows the brown resin-like materials and starch grains in IV section by using I2-KI staining; D-2 shows the brown resin-like materials and polysaccharides in IV section by using PAS staining; D-3 shows the brown resinlike materials and nuclei in IV section by using DAPI staining. White arrowheads indicate nuclei; red arrowheads indicate brown resin-like materials; blue arrowheads indicate starch grains; black arrowheads indicate polysaccharides. Scale bars = 100 μm. (B) A diagram of distribution patterns of storage starch, polysaccharides, brown resin-like materials and nuclei in different layers of wounded wood. DL, the decay layer; DA, the decay-agarwood transition layer; AL, the agarwood layer; TL, the agarwood-normal transition layer; NL, the normal layer. Label I shows the site of micrographs of A1~A3 in Fig. S1A; Label II shows the site of micrographs of B1~B3 in Fig. S1A; Label III shows the site of micrographs of C1~C3 in Fig. S1A; Label IV shows the site of micrographs of D1~D3 in Fig. S1A.

Results
(2) Description of the different panels in Fig. 2 must be improved. This figure includes many data, that would be relevant for conclusions in the article. I found panels D unnecessary and could be removed. Panel A requires a more detailed explanation. Not clear what are the differences between the different graphs in this panel. Thus, it is difficult to reach a conclusion. Thanks for the constructive advice. We removed panel D to Fig. S4. We also added the explanation of Fig.2A in the manuscript, and reviewed the legend of this figure as follows. Manuscript L152-158. The alpha diversity of fungal species observed was significantly higher in DL compared with the other four layers (P<0.05) ( Fig. 2A and Table S3). The community richness indices, including ACE, Chao 1, and PD whole tree, indicated that the three layers containing agarwood resin (DA, AL, TL) possessed the lowest richness. In contrast, DL possessed the richest fungal community ( Fig. 2A and Table S3). The Shannon index indicated the lowest fungal diversity in AL and TL, suggesting that the fungal specificities of AL and TL were strong ( Fig. 2A and Table S3). The legend of Figure 2  (Table   S3). Significant differences (p-value < 0.05) across different compartments were indicated with lowercase letters (a, significant difference between DL and the other layers using Tukey analysis, p value < 0.05; b, significant difference between DA and the other layers using Tukey analysis, p value < 0.05; c. significant difference between AL and the other layers using Tukey analysis, p value < 0.05; d. significant difference between TL and the other layers using Tukey analysis, p value < 0.05; e. significant difference between NL and the other layers using Tukey analysis, p value < 0.05). Significant differences (p value < 0.01) across different compartments were indicated with uppercase letters (A, significant difference between DL and the other layers using Tukey analysis, p value < 0.01; B, significant difference between DA and the other layers using Tukey analysis, p value < 0.01; C. significant difference between AL and the other layers using Tukey analysis, p value < 0.01; D. significant difference between TL and the other layers using Tukey analysis, p value < 0.01; E. significant difference between NL and the other layers using Tukey analysis, p value < 0.01). DL, decay layer; DA, decay-agarwood transition layer; AL, agarwood layer; TL, agarwood-normal transition layer; NL, normal layer. (B) Unconstrained PCoA (for principle coordinates PCo1 and PCo2) with weighted UniFrac distance depicting the fungal species composition structure differences among different layers of agarwood. (C) LEfSe generated the taxonomic cladogram showing marker fungal species of each layer of agarwood. Each dot represents one taxon, with sizes indicating average abundances. The color of dots represents taxon marker belongings of groups.
Phaeoacremonium rubrigenum was labeled with red color, which was the biomarker of the TL.
(3) Fig. 3F. Please indicate in the legend to this figure what layers were analyzed in the lower panels. Thanks for the kind advice. We have added the lower panels in Fig. 3F and the corresponding figure legend. Fig. 3 (F) Comparing relative content of total sesquiterpene and chromone accumulation differences in one-year-old A. sinensis seedlings after treatment with six other fungal species for 30 days, including L. theobromae (Lth), T. harzianum (Tha), T. atroviride (Tat), C. cladosporioides (Ccl), C. parahalotolerans (Cpa) and Penicillium janthinellum (Pja). f1: Culture characteristics of the above six fungal species isolated from agarwood on potato dextrose agar (PDA) medium. f2: Pie charts showing the relative abundance of the above fungal genera in each agarwood layer, including Lasiodiplodia sp., Trichoderma sp., Cladosporium sp., Penicillium sp. f3: Comparing the total sesquiterpene content in one-year-old A. sinensis seedlings after treatment using the holing technique with P. rubrigenum (Pmr) and other isolated fungi (Lth, Tha, Tat, Ccl, Cpa, Pja) for 30 days. n = 4. ** p value<0.01. f4: Comparing the total chromone content in one-year-old A. sinensis seedlings after treatment using the holing technique with P. rubrigenum (Pmr) and other isolated fungus (Lth, Tha, Tat, Ccl, Cpa, Pja) for 30 days. n = 4. ** p value<0.01.
(5) Authors need to revise the English to correct some misspelling words. Thanks for the reviewer's advice. We have reviewed the whole manuscript for wrong spellings, and we also searched for help from Editage for further polishing the manuscript.

Reviewer #2 (Comments for the Author):
In this manuscript, Liu et al. carried out extensive -omics-based studies to understand the interactions between Pm. rubrigenum and A. sinensis. The study itself is interesting to some extent but needs to be significantly developed before it can be accepted by Microbiology Spectrum. There are some places in the manuscript that are confusing to read and need further polishing.
(1) A key question that was not answered in this manuscript, but is generally critical in the plant-microbe interactions, is to determine the compounds of interest are synthesized by which party. The authors have carried out extensive omics studies to show that sesquiterpene production is enhanced in A. sinensis, but it will also be helpful to investigate if these terpene-related genes are in Pm. rubrigenum. There are two potential scenarios that could be interesting: i) Pm. rubrigenum provides precursors to facilitate the production of sesquiterepen in A. sinensis, or ii) Pm. rubrigenum contains the terpene synthase genes. Therefore, in Line 185-190, the control experiment to conclude that Pm. rubrigenum was key to sesquiterpene production was not conclusive. It is likely that Pm. rubrigenum itself may produce these sesquiterpenes in the absence of A. sinensis seedlings. Thanks for the reviewer's insightful advice. Just as the reviewer said, the sesquiterpenes might be produced by P. rubrigenum. Actually, we first thought P. rubrigenum itself might produce these sesquiterpenes in the absence of A. sinensis. Thus, we sequenced the genome of P. rubrigenum in the year 2018 (unpublished data). However, we didn't find any terpene synthase genes in the genome of P.
rubrigenum. Thus, we thought P. rubrigenum could not produce these sesquiterpenes, but induce A. sinensis seedlings to produce sesquiterpenes. To examine this thought, the volatile compounds of P. rubrigenum thalli and fermentation both were tested by GC-MS in this study (Fig. S6). No any sesquiterpenes could be detected. FPP, which is the precursors of sesquiterpenes, could also not be detected. Thus, it should be the A. sinensis seedling to produce agarwood sesquiterpene instead of P. rubrigenum. Thus, we mainly focused on addressing the mechanism of the induction of P. rubrigenum to its host A. sinensis. rubrigenum hyphae and fermentation liquor. n=3. There is no any sesquiterpenes and FPP detected.
(2) In Section 2.4, the proteome analysis of A. sinensis: it is unclear how the sampling of A. sinensis was carried out. Was A. sinensis one-year seedlings? Which layer (AL, NL, or TL) was examined with proteomics? Thanks for the advice. The proteome analysis of A. sinensis was carried out by using the A. sinensis calli. The reason why we choose the A. sinensis calli to detect the proteome analysis is that the callus system of A. sinensis is more stable than the other systems. We have further revised this section.
(3) There are some grammatical errors throughout the manuscript and are not limited to the following: ----Line 30, "concerning" should be "for"; Revised.

Revised.
We have revised the grammar of our manuscript thoroughly by ourselves and also by Editage for polishing the manuscript. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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